This invention relates to the fields of radiolabeled proteins, radioimmunotherapy and immunodiagnostics and, more particularly, to methods for labeling antibodies and fragments thereof with radiometals.
The attachment of radiometals to proteins, especially antibodies and antibody fragments, results in the formation of new radiodiagnostic and radiotherapeutic agents. The performance of the radiometal-protein conjugates depends on a number of factors, such as the stability of the radiometal-protein bond in the conjugates and the ability of the conjugates to localize to the target tissue. Clearly, since it is desired that radioactive agent not accumulate in any but the target tissues, the performance of such agents also depends in great part on the extent and rate at which the agents are eventually cleared from non-target tissue.
The use of monoclonal antibodies and antibody fragments in protein-radiometal conjugates should ideally provide an efficient means of localizing such conjugates to target tissue. Monoclonal antibodies are highly specific and can be used, for example, for imaging specific target sites or as vehicles to deliver other substances to such target sites. In recent years, numerous antibodies have been developed with affinity for targets such as atherosclerotic tissue, fibrinogen, myosin, and tumors, to name just a few, and work in this area continues.
Proteins and antibodies have been shown to form stable bonds to radiometals by the use of bifunctional coupling agents. The bifunctional agent is selected such that it is capable of binding radiometals by chelation and also form a stable linkage to the protein. Thus, the protein or antibody is bound to the radiometal through the bifunctional coupling agent. For example, diethylenetriaminepentaacetic acid (DTPA) has been conjugated onto an antimyosin antibody, and the protein-bound DTPA used to chelate indium-111 (Khaw, et al., Science, 209, 295-97 (1980). See also Krejcarek, et al., Biochem. Biophys. Res. Comm., 77, 581-85 (1977) and Childs, R. L. and Hnatowich, D. J., J. Nucl. Med., 26, 293 (1985)). This approach has also been used where particular diaminodithiol and diamidedithiol chelating agents have been coupled to antibodies (Fritzberg, et al., J. Nucl. Med., 27, 957-58 (1986) and Eary, J., et al., J. Nucl Med., 28, 650-51 (1987)). Chelated radiometals and bifunctional coupling agents have been linked to proteins by lysyl side chain amino groups (EPO Publication No. 188, 256). Chelators have also been site-selectively attached to oxidized antibody carbohydrate moieties (EPO Publication No. 173,629, U.S. Pat. No. 4,671,958). Chelators can also be attached by reaction with free sulfhydryl groups (U.S. Pat. No. 4,659,839, U.S. Pat. No. 4,671,958 and EPO Publication No. 173, 629).
Although means have been found for preparing stable protein-radiometal conjugates which effectively localize to target tissue, the dosage of such conjugates to non-target tissue is a factor which limits the dosage at which the conjugates may be used. The clearance of radiometallated antibodies by the use of a cleavable bifunctional coupling agent has been addressed. DTPA derivatives containing cleavable functionalities have been used to form conjugates between indium-111 and antibodies. This composition has an increased clearance rate in mice (Quadri, S. M., et al., J. Nucl. Med., 27, 959 (1986), Yokoyama, K., et al., J. Nucl. Med., 28, 572 (1987) and Paik, C. H., et al., J. Nucl. Med., 29, 889 (1988)). The bifunctional agent was linked to the antibodies by lysyl side chain amino groups. The antibody-linked chelator was labeled with indium-111, and purification was required prior to use.
Radiometals other than indium are particularly well-suited for use in immunodiagnostic and immunotherapeutic procedures. Technetium-99m, for example, is one such radiometal because of its nuclear properties (single photon energy of 140 KeV, a half-life of about 6 hours) and ready availability. There is therefore a need for protein-radiometal conjugates using radiometals such as technetium which are more readily cleared from non-target tissues than conjugates previously disclosed. There is also a need for such conjugates which can be prepared by simple methods e.g., by methods not requiring purification steps and which do not decrease the affinity of the radiolabeled protein.